During this reporting period we have continued to explore the role of the histone methyltransferases EZH1 and EZH2 in the biology of mammary epithelium. We have now generated mice that lack either the Ezh1 gene or the Ezh2 gene specifically in mammary epithelium. We have determined that neither EZH1 nor EZH2 are required for mammary development, suggesting that these enzymes can compensate for each other. This is supported by the observation that H3K27me3 levels are grossly normal in the absence of either methyltransferase. However, EZH2 appears to control the temporal progression of mammary differentiation during pregnancy. To determine whether EZH1 and EZH2 serve redundant functions we attempted to generate mice that lack both enzymes in mammary epithelium. These mice would carry two Ezh1 null alleles, two Ezh2 floxed alleles and an MMTV-cre transgene, which deletes floxed alleles in mammary stem cells and in some other cell types, including hematopoietic cells. Despite an enormous effort, screening hundreds of mice, we were unable to obtain Ezh1/2-null mice. This suggests that mice with this genotype either die perinatally or in utero. We now pursue an alternative strategy and attempt to generate primary cells that have lost EZH1 and EZH2. Towards this goal we enlisted a new technology, called conditionally reprogrammed cells (CRC) that had been developed at Georgetown University. We are currently in the process of characterizing primary mammary CRCs.